Summary Mitochondria are at the center of age-related human diseases, such as Parkinson?s, Alzheimer?s, Huntington?s diseases. With current trends toward the aging population, developing therapies for these diseases is a critical need. Protective strategies, mostly aimed at neutralizing toxic reactive oxygen species (ROS), have only been partially successful. Thus, new treatment options with independent modes of action are needed to improve the success rate of current therapies. The proposed work directly responds to this need and will provide critical insights into the regulation and function of mitochondrial quality control pathways. Eukaryotic cells have a multi-layered system dedicated to mitochondrial quality control. The initial defense consists of a network of proteolytic systems that degrade proteins that are damaged, misfolded or mislocalized, or control activities of stress responsive mitochondrial factors. Recent findings, including work in the PI?s laboratory, demonstrated that the ubiquitin (Ub)/proteasome system (UPS), through Ub-dependent degradation/control of the OMM associated proteins is critical for mitochondrial quality control. Regulation of mitochondrial fusion and fission (mitochondrial dynamics) is another principal mitochondrial quality control mechanism. Recently we proposed that stability of outer mitochondrial membrane (OMM) proteins, including mitochondrial fusion factors Mfn1 and Mfn2 is controlled by mitochondrial fission proteins Drp1 and Mff. Consistent with this, our new data indicate that previously unsuspected Ub-dependent signaling functions of Drp1 and Mff are required for this process. We also found that mitochondrial protein ubiquitination is focal and that this process is accelerated in Mff-/- and Drp1-/- cells. These and other preliminary results, suggest unappreciated mechanisms regulating and coordinating mitochondrial quality control pathways, including participation of the novel mitochondrial protein degradation intermediates and mutual dependence between mitochondrial dynamics and mitophagy. We also identified an OMM-associated E3 Ub ligase Rnf179, which according to our preliminary findings not only controls stability of OMM proteins, including Mfn1 and Mfn2, but also affects mitochondria-specific autophagy (mitophagy), a critical UPS-dependent mitochondrial quality control pathway. The principle goal of this proposal is to test the hypothesis that through Ub-dependent signaling, Drp1 and Mff control and coordinate distinct mitochondrial quality pathways, including mitochondrial fission and fusion rates and activation of mitophagy. We also anticipate defining the role and mechanism of Rnf179 with mitochondrial protein ubiquitination in the control of mitochondrial function and integration of mitochondrial quality control pathways, including Parkin- dependent and -independent mitophagy. To achieve these goals we combine biochemical, state-of-the-art imaging, and recently developed gene editing methods to address the following questions: (1) What is the Ub- dependent mechanism that mediates crosstalk between mitochondrial fission and fusion machineries, and how does it maintain balance between these two processes? (2) What is the mechanism by which Rnf179 in concert with Mff and Drp1 controls mitochondrial homeostasis? (3) What is the mechanism by which Rnf179 in concert with Mff and Drp1 complex controls mitophagy?